Abstract: This study was conducted to develop a protocol for in vitro propagation of hermaphroditic papaya (Carica papaya L.) from shoot buds. In this study, MS media modified with varying concentration of auxin and cytokinin were used to evaluate the effect of cytokinin and auxin for establishment of the appropriate medium in regeneration of carica papaya under in vitro condition. Best initiated and proliferated shoots were obtained on MS medium with 1mg/l BAP and 0.5 mg/l NAA. The highest mean number of shoots (16), highest shoot length (1.7 cm) and mean number of leaves (21) were recorded on MS medium with 1.0 mg/l BAP and 0.5 mg/l of NAA. Likewise, the minimum and maximum number of days for shoot emergence was counted on BAP at 0.5 mg/l +0.5mg/l NAA and BAP at 2.0 mg/l + 0.5 mg/l NAA respectively. The lowest number of shoots, number of leaves and shoot length was recorded as BAP was increased from 0.5 mg/l to 2.0 mg/l and at 0.5 mg/l BAP. On the other hand, the maximum number of roots (16.25) and root length (3.92 cm) were measured on shoots pretreated on MS media supplemented with 1.5 mg/l IBA. Unlike this, minimum root length and number were obtained from 3mg/l of IBA. Lowest survival capacity (40%) of seedlings were recorded during acclimatization on mixture of garden soil, sand and cow dung at the rate of 2:1:1. Overall, the study showed that BAP and NAA combination at medium level concentrations for shoot initiation and multiplication; and IBA at lower and medium level concentrations for rooting were the best basal medium for in vitro propagation of papaya taken from shoot buds; Nevertheless, further investigations are needed to minimize low survival rate of the seedlings for mass and commercial propagation of the in vitro developed protocol. Key words: BAP

IBA

NAA

Root Induction

Shoot Bud

INTRODUCTION

Shoot Initiation

Carica Papaya

Southeast Asian region [4]. Now a days it is also popularly cultivated in Ethiopia as a good source of iron, calcium, vitamins A, B, C [5-6] and the latex is also a good source of proteolytic enzymes, papain and chymopapain. It can be propagated sexually by using seeds and asexual through grafting and rooted cuttings also exists. However, the setback of propagating by seeds is the production of non-true-to-type planting materials due to the segregation of off springs at the second filial generation; the inherent heterozygosity and dioecious nature of the plant; and the seeds of open-pollinated flowers exhibit considerable variation in shape, size and flavour and susceptibility to diseases[7]. Similarly asexual reproductions are also often tedious and impractical when carried out on large scale. Therefore to minimize these problems, efficient micro propagation of papaya has

The papaya (Carica papaya L.) belongs to the small family Caricaceae, which includes 35 species placed in six genera. Among all species, 32 are dioecious, two trioecious and one monoecious [1]. The papaya is the only species of the genus Carica, also being the best known and most economically important within the family [2]. Botanically, papaya plant has three different sex types: staminate (male plant) producing staminate flower, female plant producing pistillate flower (female plants) and hermaphrodite plants producing bisexual flower [3]. It is an important fruit crop grown in the tropical and sub-tropical regions of the world. It is now widely cultivated throughout the tropical world and in the warmest parts of the subtropics and elsewhere in the

become critical for the multiplication of specific sex types of papaya [8] and in the application of genetic transformation technologies. Hence, breeding and cultivation of papaya is limited to conventional techniques in Ethiopia and no work has been conducted on in vitro propagation of papaya, this study was carried out to fill this gap by developing an efficient and reproducible propagation protocol by using shoot bud culture of papaya (Carica papaya L).

The surface sterilized and trimmed explants were cultured on a shoot initiation medium consisting of MS base (Murashige and Skoog, 1962) and BAP at (0.0, 0.5, 1, 1.5 and 2.0 mg/l) in combination with NAA at 0.5 mg/l concentration. The culture vessel was sealed with a parafilm and kept in a growth room under 16:8 h (light: dark) photoperiod regime with light intensity of 2000-2500 lux at 25 ± 2°C and 70% relative humidity (RH). Sub culturing was done by transferring the new micro plantlets in to fresh media within 2 weeks of interval. Growth performance of the shoots(number of days for shoot initiation, number of leafs, number of shoots and shoot length were recorded per a week.

MATERIALS AND METHODS The study was conducted at the Tissue Culture Research Laboratory of Plant Biotechnology Division in Mekelle Agricultural Research Center (MARC), Northern Ethiopia during the period of 2012/13.

Rooting of Shoots: The shoots with more than 1.0 to 1.8 cm in height were taken out from the jar and then placed in half strength MS media supplemented with (0, 0.75, 1.5, 2.25 and 3 mg/l) concentration of IBA. The pH was adjusted to 5.8 with 0.1% N NaOH and 0.1% N HCl prior to autoclaving at 121°C and 15 pounds per square inch (psi) for 20 min. The culture room was maintained at 25 ± 2°C temperature uniform light was provided by using florescent tubes 2000-2500 lux at 25 ± 2°C and 70 % relative humidity (RH) over a light/dark cycles of 16/8 hrs. Number of roots and root length were measured after a month of culturing.

Preparation of Plant Material: The explants were shoot buds obtained from three month old greenhouse grown hermaphroditic Carica papaya L. cv. Maradol seedlings. The shoot buds of papaya were washed thoroughly under running tap water and then treated with 3 drops of tween-80 for about 7 min with constant shaking. Subsequently the explants were transferred to laminar airflow cabinet and placed into sterilized jar. Then the explants were immersed in 0.1% HgCl2 and shaked gently for further sterilization for 5min. The explants were then washed with sterile distilled water 5 times to remove every trace of sterilants. After sterilization, the outer leaves were removed and 3 cm long shoot buds were excised and prepared for inoculation.

Acclimatization: Young rooted papaya plantlets were taken out from the glass Jars and washed with running tap water gently to remove traces of agar. Plantlets with actively growing roots were planted in to a pot having coco peat soil. After four days in the coco peat soil the plantlets were transferred to plastic pots containing a mixture of garden soil, cow dung and sand with 2:1:1 ratio and covered with polyethylene to retain high humidity. Plantlets were covered with plastic sheets and maintained in shade netted greenhouse for one month. Finally, survival capacity of the plantlets was recorded.

Culture Media Preparation and Culture Establishment: Murashige and Skoog medium were used for all the experiments consisted of the basic macro and micro nutrients and organic supplements. In all cases MS stock solutions [9] supplemented with 30 gm/l sucrose as a carbon source, 8 gm/l agar was used. BAP (0.0, 0.5, 1.0, 1.5 and 2.0 mg/l) in combination with consistent concentrations of NAA (0.5 mg/l) for initiation and proliferation of shoots were prepared. The pH was adjusted to 5.8 by addition of 0.1% N HCl and 0.1% NaOH as required. The basal medium was then poured into the Magenta culture vessels and medium was autoclaved at 15 pounds per square inch (psi) and 121°C for 20 minutes. Finally, all the autoclaved culture media were allowed to cool at room temperature and stored in culture rooms until further use.

RESULTS AND DISCUSSION Establishment and Proliferation of Shoots from Bud [H1]: As indicated in T(1), there is a significant difference (p